A novel red fluorescence dopamine biosensor selectively detects dopamine in the presence of norepinephrine in vitro

Molecular Brain

Chihiro Nakamoto¹  Tomonori Takeuchi¹  David E. Gloriam²  Kasper Harpsøe²  Yuko Fukata³  Masaki Fukata³  Yoko Tomizawa⁴  Yuhei Goto⁵  Kazuhiro Aoki⁵ 

[1] Department of Biomedicine, Aarhus University, Hoegh-Guldbergsgade 10, 8000, Aarhus C, Denmark;Danish Research Institute of Translational Neuroscience - DANDRITE, Nordic-EMBL Partnership for Molecular Medicine, Aarhus University, Hoegh-Guldbergsgade 10, 8000, Aarhus C, Denmark;Center for Proteins in Memory - PROMEMO, Danish National Research Foundation, Department of Biomedicine, Aarhus University, Hoegh-Guldbergsgade 10, 8000, Aarhus C, Denmark;Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark;Division of Membrane Physiology, Department of Molecular and Cellular Physiology, National Institute for Physiological Sciences, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Department of Physiological Sciences, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Division of Quantitative Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Quantitative Biology Research Group, Exploratory Research Center on Life and Living Systems (ExCELLS), National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Division of Quantitative Biology, National Institute for Basic Biology, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), 5-1 Higashiyama, Myodaiji-cho, 444-8787, Okazaki, Aichi, Japan;

关键词: GPCR;    Dopamine;    Norepinephrine;    Fluorescence probe;    Hippocampal neuron;   

DOI  :  10.1186/s13041-021-00882-8

来源: Springer

【 摘 要 】

Dopamine (DA) and norepinephrine (NE) are pivotal neuromodulators that regulate a broad range of brain functions, often in concert. Despite their physiological importance, untangling the relationship between DA and NE in the fine control of output function is currently challenging, primarily due to a lack of techniques to allow the observation of spatiotemporal dynamics with sufficiently high selectivity. Although genetically encoded fluorescent biosensors have been developed to detect DA, their poor selectivity prevents distinguishing DA from NE. Here, we report the development of a red fluorescent genetically encoded GPCR (G protein-coupled receptor)-activation reporter for DA termed ‘R-GenGAR-DA’. More specifically, a circular permutated red fluorescent protein (cpmApple) was replaced by the third intracellular loop of human DA receptor D1 (DRD1) followed by the screening of mutants within the linkers between DRD1 and cpmApple. We developed two variants: R-GenGAR-DA1.1, which brightened following DA stimulation, and R-GenGAR-DA1.2, which dimmed. R-GenGAR-DA1.2 demonstrated a reasonable dynamic range (ΔF/F0 = − 43%), DA affinity (EC50 = 0.92 µM) and high selectivity for DA over NE (66-fold) in HeLa cells. Taking advantage of the high selectivity of R-GenGAR-DA1.2, we monitored DA in presence of NE using dual-color fluorescence live imaging, combined with the green-NE biosensor GRABNE1m, which has high selectivity for NE over DA (> 350-fold) in HeLa cells and hippocampal neurons grown from primary culture. Thus, this is a first step toward the multiplex imaging of these neurotransmitters in, for example, freely moving animals, which will provide new opportunities to advance our understanding of the high spatiotemporal dynamics of DA and NE in normal and abnormal brain function.

【 授权许可】

CC BY